Myotonic Dystrophy type 2 (DM2) is an autosomal dominant, multisystemic disease, that primarily affects skeletal muscle causing skeletal muscle loss, muscle weakness and myotonia. DM2 is caused by expansion of CCTG repeats in the intron 1 of ZNF9 gene. CCTG expansion induces DM2 pathology through accumulation of RNA CCUG repeats that misregulate RNA metabolism. Muscle wasting is a major health problem in patients with DM2. The mechanisms by which CCUG repeats cause skeletal muscle wasting are not known. Our recent findings show that the mutant CCUG repeats reduce ZNF9 protein. Because ZNF9 controls synthesis of proteins of translational apparatus, its reduction in DM2 myoblasts decreases the rate of global protein synthesis. Therefore, the main hypothesis of this application is that the mutant CCUG repeats cause skeletal muscle loss through decrease of the rate of global protein synthesis.
The Aim 1 of this application will determine mechanisms by which CCUG repeats down regulate ZNF9 protein in DM2. Since CCUG repeats do not change ZNF9 mRNA levels, we will determine if CCUG repeats reduce protein synthesis of ZNF9 or if the CCUG repeats increase degradation of ZNF9. Immunofluorescent analysis revealed that, in DM2 myofibers, ZNF9 translocates to the myofiber membrane suggesting that CCUG repeats might reduce cytoplasmic ZNF9 by enhancement of its interaction with membrane proteins. Therefore, we will test if the re- distribution of ZNF9 to the membrane of DM2 myofibers causes the reduction of ZNF9 in cytoplasm. Reduction of ZNF9 in muscle biopsies from DM2 patients suggests that the rate of global protein synthesis is reduced in mature muscle in DM2 patients. Therefore, Aim 2 will determine if the mutant CCUG repeats reduce the rate of global protein synthesis in vivo causing skeletal muscle wasting. For this goal, we will utilize the DM2 mouse model, CCTG transgenic mice, which have low levels of ZNF9. ZNF9 will be normalized in CCTG TR mice and the effect of correction of ZNF9 on muscle wasting and muscle function will be examined.
In Aim 3, we propose to elucidate the mechanisms responsible for the increased stability of the mutant CCUG repeats with the goal to eliminate toxicity of CCUG repeats. We found that pure CCUG repeats are very stable. We have identified five CCUG100-binding proteins that alter activity during accumulation of the mutant CCUG repeats and in DM2 myoblasts. We hypothesize that alterations in activity of these proteins are responsible for the increased stability and toxicity of the mutant CCUG repeats. One of these proteins has been purified and determined as p68 helicase. We will elucidate the relative role of p68 and other identified CCUG100-binding proteins in the regulation of stability of the mutant CCUG repeats. The results of the proposed studies will determine the molecular mechanism of skeletal muscle loss in patients with DM2 and will help to develop approaches to degrade mutant CCUG repeats.

Public Health Relevance

Patients with Myotonic Dystrophy type 2 (DM2) suffer from skeletal muscle loss and muscle weakness. The application proposes to apply basic science expertise to outline the mechanism of muscle wasting in DM2 and to outline the mechanism controlling degradation of the mutant CCUG repeats. Our basic science approaches will provide the ground work for the development of the clinical treatment of DM2.